The applicant proposes a two aim strategy to explore molecular mechanisms of the regulation of ion-motive ATPases. Aim 1 focuses on the cardiac calcium pump (SERCA) and its regulation by phospholamban (PLB). The proposed research will use fluorescence resonance energy transfer (FRET) and chemical crosslinking to test the hypothesis that PLB binds to multiple sites on SERCA, and that these discrete binding sites mediate different functional effects. Aim 2 describes a novel high throughput assay for investigation of the binding interactions of phospholemman (PLM) with itself and its regulatory target, the sodium/potassium pump (NKA). The goal of the proposed experiments are to develop a novel peptide superinhibitor of NKA as a safer alternative to cardiac glycosides, a class of inotropic drugs used in the treatment of heart failure. The experiments described in the two Aims of this application will provide new insight into fundamental mechanisms of regulation of ion-motive ATPases, and may improve our understanding of the ion transport disorders associated with heart failure.